The peer-reviewed and funded Collaborative Projects and the Service Projects from leading hyperpolarized MR sites, that are the scientific driving force behind the HMTRC, have demonstrated a clear need for improved hyperpolarized (HP) probes and techniques, and more realistic pre-clinical models of human disease. In this TR&D project we will take advantage of our extensive experience in combining hyperpolarized MR techniques with novel NMR-compatible bioreactors to develop and provide improved pre-clinical models for hyperpolarized MR probe testing. The TR&D project also builds on our extensive experience with HP MR probe development and optimization, and the unique HP MR environment at UCSF. Specifically, in aim 1 we will develop cell and tissue culture bioreactors that have the sensitivity to reduce the amount of cells and tissues required by 10-fold, that are inexpensive to build and robust to use, and are capable of maintaining the structure and function of the primary cell and tissue cultures proposed in the Collaborative Projects for prolonged durations. In aim 2, the development and optimization of new HP probes capable of ROS quantification and probing glycolysis upstream of pyruvate will be developed and optimized. Additionally, we will further optimize multi-polarization approaches that provide a means of probing several enzymatic pathways and other physiologic properties (perfusion, pH, necrosis) in vivo, in a single MR acquisition. In aim 3, the NMR-compatible bioreactor systems, and hyperpolarized probes and methods will be adapted for the specific applications described in the Collaborative Projects. These studies will produce tailored HP MR procedures and correlative pathologic and molecular analyses to better understand and validate the HP MR biomarkers. Since the ultimate goal of this TR&D project is the dissemination of the research findings to the biomedical community, the chemistry preparation methods for HP agents, optimized polarization methods, and cell and tissue specific NMR compatible cell and tissue culture bioreactor procedures will be made available through publications, documentation on the HMTRC website, yearly symposiums, and hands-on training.